Heparan sulfate (HS) is widely expressed in animal and human tissues and has diverse roles in development, differentiation, and homeostasis. HS and other glycosaminoglycans are unbranched polymers covalently attached to the protein cores of proteoglycans, which are ubiquitously expressed as integral membrane proteins, glycerol phosphatidyl inositol-linked membrane proteins, and proteins of the extracellular matrix. The HS polymer is assembled by sequential addition of D-glucuronic acid (GlcA) alternating with N-acetyl glucosamine (GlcNAc). The chains are then modified heterogeneously and in domains by de-acetylation and sulfation of glucosamine, epimerization of GlcA residues to iduronic acid (IdoA), and sulfation of hydroxyl groups. These modifications provide specific binding sites for a variety of proteins, including cell adhesion molecules, growth factors, chemokines, and factors regulating angiogenesis and blood coagulation. Protein binding to HS may serve to sequester the protein at a particular site or to activate the protein. For example, the binding of antithrombin to a specific pentasaccharide sequence in HS results in activation of its anticoagulant activity (reviewed by Lindahl et al., 1998; Rosenberg et al., 1997).
Herpes simplex viruses (HSV) are human herpesviruses of the neurotropic alpha herpesvirus subfamily. Infections with HSV type 1 (HSV-1) and HSV type 2 (HSV-2) are highly prevalent. The usual manifestations of disease are mucocutaneous lesions of the mouth, face, eyes, or genitalia. Both HSV-1 and HSV-2 establish latent infections in neurons of peripheral ganglia and may reactivate to cause recurrent lesions. Rarely, the virus spreads to the central nervous system to cause meningitis or encephalitis (reviewed by Spear, 1993).
A number of viruses, including the human herpesviruses of the neurotropic alpha herpesvirus subfamily, use sites on HS as receptors for binding to cells (Byrnes and Griffin, 1998; Chung et al., 1998; Compton et al., 1993; Jackson et al., 1996; Shieh et al., 1992; Summerford and Samulski, 1998). Viral entry may require interactions with other cell surface receptors as well (Geraghty et al., 1998; Montgomery et al., 1996; Summerford et al., 1999; Warner et al., 1998).
Infection involves (1) virus attachment to the cell surface membrane, followed by (2) virus penetration and entry into the cells. These two steps can be experimentally dissociated. In the case of HSV-1 and HSV-2, the virus binds to cells through interactions of cell surface HS with viral envelope glycoproteins gB and/or gC (reviewed by Spear, 1993). Certain cell types, such as swine testis or Chinese hamster ovary (CHO) cells, are resistant to viral entry, even though the viral binding to these cells appears normal. Following binding, a third viral envelope glycoprotein, gD, interacts with one of multiple specific receptors, resulting in viral entry by fusion of the virion envelope with the target cell membrane. This fusion reaction requires the concerted action of three additional viral glycoproteins, gB, gH and gL (reviewed by Spear, 1993), and appears to be triggered by the binding of gD to its cognate receptors.
The human gD-binding receptors that have been identified include a member of the TNF receptor family, designated HVEM (Montgomery et al., 1996; Whitbeck et al., 1997) or herpesvirus entry protein A (HveA), and officially named TNFRSF14, and two members of the immunoglobulin superfamily (Geraghty et al., 1998; Krummenacher et al., 1998; Warner et al., 1998). The latter two proteins are related to the poliovirus receptor (CD155) (Mendelsohn et al., 1989), were originally designated poliovirus receptor-related proteins 1 (Lopez et al., 1995) and 2 (Eberlé et al., 1995), and more recently have been referred to as HveC (Geraghty et al., 1998), and HveB (Warner et al., 1998) or nectin 1 and nectin 2 (Takahashi et al., 1999), respectively. Both HveA and HveC serve as gD-binding entry receptors for wild-type HSV-1 and HSV-2 strains, whereas HveB serves as an entry receptor for only a subset of HSV strains, (Geraghty et al., 1998; Montgomery et al., 1996; Warner et al., 1998). These gD-binding cell surface receptors are expressed at different levels in various human tissues and cell lines, suggesting a specific susceptibility to HSV in certain tissues.
It has been demonstrated that heparan sulfate 3-O-sulfotransferases (3-OSTs) are present as several isoforms (i.e., 3-OST-1, 3-OST-2, 3-OST-3, 3-OST-4) that are expressed at different levels in various tissues and cells (Shworak et al., 1999). In particular, 3-OST-3 enzymes are present in two highly similar forms (3-OST-3A and 3-OST-3B). In general, different 3-OST isoforms sulfate glucosamine residues with different saccharide sequences around the modification site. For example, 3-OST-1, 3-OST-2, and 3-OST-3 generate different saccharide sequences (Liu et al., 1999a). However, the highly homologous isoforms, 3-OST-3A and 3-OST-3B, have been shown to sulfate identical saccharide sequences. The differences in sulfation of saccharide sequences are thought to correlate with differences in function. For example, heparan sulfates, which have been 3-O-sulfated by 3-OST-1, possess anticoagulant activity, however, heparan sulfates which have been 3-O-sulfated by 3-OST-2 and 3-OST-3A do not possess anticoagulant activity.
It has been shown that 3-OST-3s sulfate N-unsubstituted glucosamine residues (Liu et al., 1999b). However the majority of the glucosamine residues in HS are present either in the form of N-acetylated or N-sulfated glucosamine, and N-unsubstituted glucosamine residues are relatively rare. In fact, N-unsubstituted glucosamine residues constitute only 1-7% of total glucosamine residues (Lindahl et al., 1998). The rarity of the saccharide sequences 3-O-sulfated by the 3-OST-3s, the multiplicity of the 3-OST-3 genes in human genome, and the correlation between sulfation and function, suggest an important biological role of 3-OST-3 modified HS.